Otx2andGbx2are required for refinement and not induction of mid-hindbrain gene expression

Development ◽  
2001 ◽  
Vol 128 (24) ◽  
pp. 4979-4991 ◽  
Author(s):  
James Y. H. Li ◽  
Alexandra L. Joyner
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Mouse Embryos ◽  
Anterior Region ◽  
Spatial Domains ◽  
Cerebellum Development

Otx2 and Gbx2 are among the earliest genes expressed in the neuroectoderm, dividing it into anterior and posterior domains with a common border that marks the mid-hindbrain junction. Otx2 is required for development of the forebrain and midbrain, and Gbx2 for the anterior hindbrain. Furthermore, opposing interactions between Otx2 and Gbx2 play an important role in positioning the mid-hindbrain boundary, where an organizer forms that regulates midbrain and cerebellum development. We show that the expression domains of Otx2 and Gbx2 are initially established independently of each other at the early headfold stage, and then their expression rapidly becomes interdependent by the late headfold stage. As we demonstrate that the repression of Otx2 by retinoic acid is dependent on an induction of Gbx2 in the anterior brain, molecules other than retinoic acid must regulate the initial expression of Otx2 in vivo. In contrast to previous suggestions that an interaction between Otx2- and Gbx2-expressing cells may be essential for induction of mid-hindbrain organizer factors such as Fgf8, we find that Fgf8 and other essential mid-hindbrain genes are induced in a correct temporal manner in mouse embryos deficient for both Otx2 and Gbx2. However, expression of these genes is abnormally co-localized in a broad anterior region of the neuroectoderm. Finally, we find that by removing Otx2 function, development of rhombomere 3 is rescued in Gbx2–/– embryos, showing that Gbx2 plays a permissive, not instructive, role in rhombomere 3 development. Our results provide new insights into induction and maintenance of the mid-hindbrain genetic cascade by showing that a mid-hindbrain competence region is initially established independent of the division of the neuroectoderm into an anterior Otx2-positive domain and posterior Gbx2-positive domain. Furthermore, Otx2 and Gbx2 are required to suppress hindbrain and midbrain development, respectively, and thus allow establishment of the normal spatial domains of Fgf8 and other genes.

72 HISTONE METHYLATION AND GENE EXPRESSION PATTERNS IN PRE-IMPLANTATION EMBRYOS DERIVED FROM INTRA- AND INTER-SPECIES NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 144
Author(s):  
W. Shi ◽  
F. Yang ◽  
E. Wolf ◽  
V. Zakharchenko
Keyword(s):  
Gene Expression ◽  
Histone Methylation ◽  
Methylation Level ◽  
Dynamic Change ◽  
Expression Patterns ◽  
Mouse Embryos ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Rabbit Embryos

The differential epigenetic changes in embryos from different species provide a model to study how the nucleus from one species interacts with cytoplasm from another species. In this study we examined histone methylation at lysine 9 of histone 3 (K9H3) and lysine 20 of histone H4 (K20H4) and the expression levels of three early development-related genes (Oct-4, Hsp 70.1 and Hprt) in individual intra- and inter-species cloned and control embryos at the 1-, 2-, 4- and 8-cell stages. Mouse fetal fibroblast (MFF) nuclei were transferred into mouse, bovine, or rabbit oocytes. As control, we used in vivo derived (mouse and rabbit) or in vitro-produced (bovine) embryos. Histone methylation was detected by anti-MeK9H3 and anti-MeK20H4 antibodies. Gene expression analysis was performed using a quantitative RT-PCR technique (Daniels et al. 2000 Biol. Reprod. 63, 1034-1040). Data were analyzed by Student's t-test. No embryos from inter-species cloning (MFF-bovine and MFF-rabbit) survived beyond the 8-12 cell stage. MFF-mouse and MFF-bovine embryos exhibited demethylation of K9H3 and K20H4 at the 2-cell stage and the methylation level was increased at the 4-cell stage, but no demethylation was observed at the 2-cell stage of MFF-rabbit embryos and the methylation level in these embryos was significantly higher than that of in vivo rabbit embryos. The level of Oct-4 mRNA was low at the 1- and 2-cell stages of in vivo mouse embryos and increased at the 8-cell stage. No significant increase in Oct-4 transcript was detected at the 8-cell stage of inter-species cloned embryos. The expression of Hsp 70.1 in in vivo mouse embryos was increased at the 2-cell stage and decreased to a level similar to that in the zygote at the 8-cell stage. In cloned embryos, Hsp 70.1 transcripts were also increased at the 2-cell stage, but there was no significant decrease of Hsp70.1 mRNA abundance at the 8-cell stage of inter-species embryos as compared to the corresponding 2-cell stage. For MFF-mouse embryos, Hsp 70.1 expression was increased at the 2-cell stage, but at the 8-cell stage the transcript level was at the level similar to that in inter-species clones. Hprt expression was increased at the 8-cell stage of in vivo mouse embryos. The dynamic change of Hprt transcript in MFF-mouse embryos was not significantly different from that of in vivo mouse embryos, but no significant change of Hprt expression occurred in the development of MFF-bovine and MFF-rabbit embryos. Differential epigenetic characteristics of mouse somatic nucleus after transfer into oocytes from different species suggest the existence of incompatibilities of nuclear-cytoplasm interaction between distantly related species. This abnormal interaction at the time of genome activation may affect normal development. This work was supported by the Bayerische Forschungsstiftung and by Therapeutic Human Polyclonals, Inc.

scholarly journals A Retinoid-Resistant Acute Promyelocytic Leukemia Subclone Expresses a Dominant Negative PML-RARα Mutation

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4282-4289 ◽  
Author(s):  
Wenlin Shao ◽  
Laura Benedetti ◽  
William W. Lamph ◽  
Clara Nervi ◽  
Wilson H. Miller
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Ligand Binding ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Regulation Of Gene Expression ◽  
Promyelocytic Leukemia ◽  
Dominant Negative

Abstract The unique t(15; 17) of acute promyelocytic leukemia (APL) fuses the PML gene with the retinoic acid receptor α (RARα) gene. Although retinoic acid (RA) inhibits cell growth and induces differentiation in human APL cells, resistance to RA develops both in vitro and in patients. We have developed RA-resistant subclones of the human APL cell line, NB4, whose nuclear extracts display altered RA binding. In the RA-resistant subclone, R4, we find an absence of ligand binding of PML-RARα associated with a point mutation changing a leucine to proline in the ligand-binding domain of the fusion PML-RARα protein. In contrast to mutations in RARα found in retinoid-resistant HL60 cells, in this NB4 subclone, the coexpressed RARα remains wild-type. In vitro expression of a cloned PML-RARα with the observed mutation in R4 confirms that this amino acid change causes the loss of ligand binding, but the mutant PML-RARα protein retains the ability to heterodimerize with RXRα and thus to bind to retinoid response elements (RAREs). This leads to a dominant negative block of transcription from RAREs that is dose-dependent and not relieved by RA. An unrearranged RARα engineered with this mutation also lost ligand binding and inhibited transcription in a dominant negative manner. We then found that the mutant PML-RARα selectively alters regulation of gene expression in the R4 cell line. R4 cells have lost retinoid-regulation of RXRα and RARβ and the RA-induced loss of PML-RARα protein seen in NB4 cells, but retain retinoid-induction of CD18 and CD38. Thus, the R4 cell line provides data supporting the presence of an RARα-mediated pathway that is independent from gene expression induced or repressed by PML-RARα. The high level of retinoid resistance in vitro and in vivo of cells from some relapsed APL patients suggests similar molecular changes may occur clinically.

scholarly journals Retinoic acid activates interferon regulatory factor-1 gene expression in myeloid cells

Blood ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 114-123 ◽  
Author(s):  
S Matikainen ◽  
T Ronni ◽  
M Hurme ◽  
R Pine ◽  
I Julkunen
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Growth Inhibition ◽  
Molecular Mechanisms ◽  
Gene Activation ◽  
Oligoadenylate Synthetase ◽  
Nb4 Cells ◽  
Drug Of Choice

All-trans-retinoic acid (ATRA) is the drug of choice in the treatment of acute promyelocytic leukemia (APL). ATRA induces both in vitro and in vivo differentiation of APL cells into mature granulocytes. However, the molecular mechanisms involved in ATRA-dependent growth inhibition and cellular differentiation are not presently understood. The NB4 cell line, which is derived from the bone marrow of a patient with APL during relapse, can be used as a model system to study the growth and differentiation of APL cells. Because interferon (IFN) regulatory factors (IRF-1 and IRF-2) and other IFN-inducible gene products regulate cell growth, we analyzed the effects of ATRA on the expression of these genes. We show that ATRA directly activates IRF-1 gene expression, followed by activation of IRF-2 and 2′–5′ oligoadenylate synthetase (OAS) gene expression with slower kinetics. In addition to NB4 cells, ATRA also activated IRF-1 gene expression in HL-60, U937, and THP-1 cells, which all respond to ATRA by growth inhibition. A more than additive increase in IRF-1 gene expression was seen with ATRA and IFN-gamma in NB4 cells. ATRA did not activate nuclear factor kappa B or signal transducer and activator of transcription (STAT) activation pathways, suggesting that an alternate mechanism is involved in IRF-1 gene activation. The ATRA-induced expression of IRF-1, an activator of transcription and repressor of transformation, may be one of the molecular mechanisms of ATRA-induced growth inhibition, and the basis for the synergistic actions of ATRA and IFNs in myeloid leukemia cells.

scholarly journals Effect of Retinoic Acid on Morphogenesis of Inner Ears in Mouse Embryos; an In Vivo Study

1994 ◽  
Vol 53 (Suppl-10) ◽  
pp. 21-23
Author(s):  
Toshihisa Hatta ◽  
Jun Udagawa ◽  
Ryuju Hashimoto ◽  
Hiroyuki Naora ◽  
Osamu Tanaka
Keyword(s):  
Retinoic Acid ◽  
Mouse Embryos ◽  
In Vivo Study

Exogenous retinoic acid rapidly induces anterior ectopic expression of murine Hox-2 genes in vivo

Development ◽  
1992 ◽  
Vol 116 (2) ◽  
pp. 357-368 ◽  
Author(s):  
R.A. Conlon ◽  
J. Rossant
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Ectopic Expression ◽  
Teratogenic Effects ◽  
Altered Expression ◽  
Early Embryos ◽  
Specific Manner ◽  
Maternal Administration

Exogenous retinoic acid (RA) has teratogenic effects on vertebrate embryos and alters Hox-C gene expression in vivo and in vitro. We wish to examine whether RA has a role in the normal regulation of Hox-C genes, and whether altered Hox-C gene expression in response to RA leads to abnormal morphology. The expression of 3′ Hox-2 genes (Hox-2.9, Hox-2.8, Hox-2.6 and Hox-2.1) and a 5′ gene (Hox-2.5) were examined by whole-mount in situ hybridization on embryos 4 hours after maternal administration of teratogenic doses of RA on embryonic day 7 to 9. The expression of the 3′ Hox-2 genes was found to be ectopically induced in anterior regions in a stage-specific manner. The Hox-2.9 and Hox-2.8 genes were induced anteriorly in the neurectoderm in response to RA on day 7 but not at later stages. Expression of Hox-2.6 and Hox-2.1 was ectopically induced anteriorly in neurectoderm in response to RA on day 8. Hox-2.1 remained responsive on day 9, whereas Hox-2.6 was no longer responsive at this stage. The expression of the 5′ gene Hox-2.5 was not detectably altered at any of these stages by RA treatments. We also examined the response of other genes whose expression is spatially regulated in early embryos. The expression of En-2 and Wnt-7b was not detectably altered by RA, whereas RAR beta expression was induced anteriorly by RA on day 7 and 8. Krox-20 expression was reduced in a stage- and region-specific manner by RA. The ectopic anterior expression of Hox-2.8 and Hox-2.9 induced by RA on day 7 was persistent to day 8, as was the altered expression of Krox-20. The altered pattern of expression of these genes in response to RA treatment on day 7 may be indicative of a transformation of anterior hindbrain to posterior hindbrain, specifically, a transformation of rhombomeres 1 to 3 towards rhombomere 4 identity with an anterior expansion of rhombomere 5. The ectopic expression of the 3′ Hox-2 genes in response to RA is consistent with a role for these genes in mediating the teratogenic effects of RA; the rapid response of the Hox-C genes to RA is consistent with a role for endogenous RA in refining 3′ Hox-C gene expression boundaries early in development.

scholarly journals Retinoic acid activates interferon regulatory factor-1 gene expression in myeloid cells

Blood ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 114-123 ◽  
Author(s):  
S Matikainen ◽  
T Ronni ◽  
M Hurme ◽  
R Pine ◽  
I Julkunen
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Growth Inhibition ◽  
Molecular Mechanisms ◽  
Gene Activation ◽  
Oligoadenylate Synthetase ◽  
Nb4 Cells ◽  
Drug Of Choice

Abstract All-trans-retinoic acid (ATRA) is the drug of choice in the treatment of acute promyelocytic leukemia (APL). ATRA induces both in vitro and in vivo differentiation of APL cells into mature granulocytes. However, the molecular mechanisms involved in ATRA-dependent growth inhibition and cellular differentiation are not presently understood. The NB4 cell line, which is derived from the bone marrow of a patient with APL during relapse, can be used as a model system to study the growth and differentiation of APL cells. Because interferon (IFN) regulatory factors (IRF-1 and IRF-2) and other IFN-inducible gene products regulate cell growth, we analyzed the effects of ATRA on the expression of these genes. We show that ATRA directly activates IRF-1 gene expression, followed by activation of IRF-2 and 2′–5′ oligoadenylate synthetase (OAS) gene expression with slower kinetics. In addition to NB4 cells, ATRA also activated IRF-1 gene expression in HL-60, U937, and THP-1 cells, which all respond to ATRA by growth inhibition. A more than additive increase in IRF-1 gene expression was seen with ATRA and IFN-gamma in NB4 cells. ATRA did not activate nuclear factor kappa B or signal transducer and activator of transcription (STAT) activation pathways, suggesting that an alternate mechanism is involved in IRF-1 gene activation. The ATRA-induced expression of IRF-1, an activator of transcription and repressor of transformation, may be one of the molecular mechanisms of ATRA-induced growth inhibition, and the basis for the synergistic actions of ATRA and IFNs in myeloid leukemia cells.

scholarly journals MicroRNA-124-3p Plays a Crucial Role in Cleft Palate Induced by Retinoic Acid

2021 ◽  
Vol 9 ◽  
Author(s):  
Hiroki Yoshioka ◽  
Yurie Mikami ◽  
Sai Shankar Ramakrishnan ◽  
Akiko Suzuki ◽  
Junichi Iwata
Keyword(s):  
Cell Proliferation ◽  
Retinoic Acid ◽  
Cleft Palate ◽  
Cleft Lip ◽  
Specific Inhibitor ◽  
Mouse Embryos ◽  
Congenital Birth Defects ◽  
Excessive Intake

Cleft lip with/without cleft palate (CL/P) is one of the most common congenital birth defects, showing the complexity of both genetic and environmental contributions [e.g., maternal exposure to alcohol, cigarette, and retinoic acid (RA)] in humans. Recent studies suggest that epigenetic factors, including microRNAs (miRs), are altered by various environmental factors. In this study, to investigate whether and how miRs are involved in cleft palate (CP) induced by excessive intake of all-trans RA (atRA), we evaluated top 10 candidate miRs, which were selected through our bioinformatic analyses, in mouse embryonic palatal mesenchymal (MEPM) cells as well as in mouse embryos treated with atRA. Among them, overexpression of miR-27a-3p, miR-27b-3p, and miR-124-3p resulted in the significant reduction of cell proliferation in MEPM cells through the downregulation of CP-associated genes. Notably, we found that excessive atRA upregulated the expression of miR-124-3p, but not of miR-27a-3p and miR-27b-3p, in both in vivo and in vitro. Importantly, treatment with a specific inhibitor for miR-124-3p restored decreased cell proliferation through the normalization of target gene expression in atRA-treated MEPM cells and atRA-exposed mouse embryos, resulting in the rescue of CP in mice. Taken together, our results indicate that atRA causes CP through the induction of miR-124-3p in mice.

scholarly journals Noise modulation in retinoic acid signaling sharpens segmental boundaries of gene expression in the embryonic zebrafish hindbrain

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Julian Sosnik ◽  
Likun Zheng ◽  
Christopher V Rackauckas ◽  
Michelle Digman ◽  
Enrico Gratton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Fluorescence Lifetime Imaging ◽  
Dependent Manner ◽  
Cellular Mechanisms ◽  
Concentration Dependent Manner ◽  
Temporal Noise ◽  
The Face ◽  
Intracellular Binding

Morphogen gradients induce sharply defined domains of gene expression in a concentration-dependent manner, yet how cells interpret these signals in the face of spatial and temporal noise remains unclear. Using fluorescence lifetime imaging microscopy (FLIM) and phasor analysis to measure endogenous retinoic acid (RA) directly in vivo, we have investigated the amplitude of noise in RA signaling, and how modulation of this noise affects patterning of hindbrain segments (rhombomeres) in the zebrafish embryo. We demonstrate that RA forms a noisy gradient during critical stages of hindbrain patterning and that cells use distinct intracellular binding proteins to attenuate noise in RA levels. Increasing noise disrupts sharpening of rhombomere boundaries and proper patterning of the hindbrain. These findings reveal novel cellular mechanisms of noise regulation, which are likely to play important roles in other aspects of physiology and disease.

260 COMPARISON OF HOUSEKEEPING GENE EXPRESSION IN INDIVIDUAL 8-CELL STAGE MOUSE EMBRYOS PRODUCED UNDER DIFFERENT CONDITIONS

2007 ◽  
Vol 19 (1) ◽  
pp. 246
Author(s):  
A. Baji Gal ◽  
S. Mamo ◽  
S. Bodo ◽  
A. Dinnyes
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Standard Error ◽  
Real Time Pcr ◽  
Housekeeping Gene ◽  
Mouse Embryos ◽  
Cell Stage ◽  
The Mean

Real-time PCR has the potential to accurately quantify the mRNA level of selected genes in single cells and individual pre-implantation-stage embryos. The goal of our study was to examine the variations in gene expression within individual embryos of the same stage and between embryos of the same stage but from different sources. In our study, we determined expression level of the 7 most commonly used housekeeping genes in 8-cell-stage mouse embryos produced under different culture conditions. Messenger RNA of 6 embryos each that was derived in vivo, or cultured in vitro from the zygote stage, or derived from oocytes activated parthenogenetically and developed in vitro were extracted individually followed by reverse transcription into cDNA. Optimized real-time PCR was performed for cytoplasmic beta-actin (Actb), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), H2A histone family, member Z (H2afz), hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1), ubiquitin C (Ubc), peptidylprolyl isomerase A (cyclophilin A) (Ppia), and eukaryotic translation elongation factor 1 epsilon 1 (Eef1e1) genes. The results were analyzed, and the percentage standard error of the mean relative expression value was compared for all genes. All 7 genes were presented above the detection limit in all samples. One or two individual embryos showed 2- to 4-fold higher mRNA levels than the average for all genes in the group. The embryos cultured in vitro showed much higher expression levels of H2afz, Ppia, and Eef1e1 genes than those in the in vivo group. The parthenogenetic group was similar to the in vivo group in expression of Actb, H2afz, Hprt, and Eef1e1 genes, but showed significant differences (P < 0.05; Student's t-test) compared to the in vitro group (Table 1). The percent standard error of the mean decreased gradually as the number of samples was increased. The 6 individual embryos in similar groups showed relatively low variability compared to embryos at similar stage but produced in different conditions. Interestingly, the parthenogenetic embryos showed a level of gene expression comparable to that of the in vivo ones, notwithstanding their culture in vitro. In conclusion, morphological observations and similarity in developmental stage alone cannot guarantee the uniformity of embryo samples, and a minimum of 4–6 replicates per treatment is needed. Moreover, we showed that culture condition itself has an effect on housekeeping gene expression, which, if neglected, might result in misinterpretation of data. Table 1.Relative expression values of the different culture groups (mean ±SE; n =6 embryos) This work was supported by EU FP6 (MEXT-CT-2003-509582 and 518240), Wellcome Trust (Grant No. 070246), and Hungarian National Science Fund (OTKA) (Grant No. T046171).

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